Studies on antimalarial activity and Nano-formulations of Annona Senegalensis Leaf fractions

Abstract

The increasing incidence of drug resistance in malaria for the existing antimalarials boost the need for new antimalarial drugs. Plants and their active compounds with antiplasmodial activity are promising source of new antimalarial compounds. The aim of the present research was to investigate the antiplasmodial activity of leaves of a traditionally used antimalarial plant Annona Senegalensis of Nigerian origin. The project used the bioassay guided approach to fractionate the phytoconstituents of leaf extract of Annona Senegalensis and examine their antiplasmodial properties. The research also aimed to formulate a suitable dosage form of the active fraction of the leaf extract for oral delivery.
The successive extraction method was employed using solvents of varied polarities ranging from polar to nonpolar solvents such as methanol, ethanol, butanol, ethyl acetate, chloroform, diethyl ether and hexane. Chloroform (CHASE) and diethyl ether extract showed the maximum in vitro antiplasmodial activity when tested at three concentration levels (100, 33.3 and 11.1 μg/ml) using SYBR Green l fluorescence assay against of P. falciparum (Dd2) chloroquine resistant strain. Suitable analytical reverse phase high-Performance liquid chromatography (HPLC) methods were developed and optimised for mobile phase gradient elution and detection wavelengths for fractionation of phytoconstituents from these extracts. For diethyl ether extract, initial elution monitored at 254nm and later switching to 400nm allowed collection of maximum constituents, while elution monitored at 400nm allowed detection of maximum peaks for CHASE. Total 10 fractions were collected from diethyl ether extract and 21 fractions were collected from CHASE by semi preparative HPLC fractionation. Out of all these fractions, fraction 13 obtained from fractionation of CHASE showed antiplasmodial activity with IC50 of 30.03 μg/ml against of P. falciparum (Dd2). Fraction 13 was further fractionated using semi preparative HPLC and an optimised mobile phase with gradient elution allowed collection of well separated five sub fractions within run time of 27 minutes. The sub fraction 4D revealed IC50 of 14.14 μg/ml when assayed using P. falciparum growth inhibition assay. NMR of the 4D revealed a compound with scaffolding that has an alkyl (methyl), a carboxylic acid, an amide group (NH), and a 1,2,3,4-tetrasubstituted benzene ring typical of flavonoids along with diethylene glycol dibenzoate which is a natural plasticiser.
Two lipid nanocarrier systems, self-emulsifying drug delivery system (SNEDDS) and nanostructured lipid carrier (NLCs) were considered for formulation of active subfraction 4D to improve its biopharmaceutical properties. 4D was formulated as type IIIB SNEDDS with Transcutol P: Caproyl 90 ratio 3:1 (6 parts) as oil phase and Tween 80: Labrasol ratio 2:1 (4 parts) as surfactant: cosurfactant mix., which when dispersed in water resulted in uniform nanoemulsion with particle size of 16.79 nm, low PDI 0.252 and zeta potential of -19.4 mV indicating good stability. The NLCs were formulated using Dynasan 118 (1%), as solid lipid and Miglyol 812 (0.5%) as liquid lipid to yield nanodispersion with particle size of 106.2nm, PDI of 0.199 and zeta potential of -3.31. The 4D loaded SNEDDS showed IC50 of 0.0303 μg/ml which was markedly enhanced as compared to IC50 of native 4D (14.14 μg/ml). However blank SNEDDS also showed IC50 0.0273 μg/ml which calls for further in vivo investigation to ascertain the merit of 4D SNEDDS. 4D loaded NLCs showed lower IC50 of 11.81 (μg/ml) with 22.2% increase in antiplasmodial activity as compared to 4D alone and comparable to that of chloroquine loaded NLCs. CHASE and its fraction 13 and subfraction 4D showed time and concentration dependent antiproliferation activity on HepG2 and HEK 293 cells. CHASE and fraction 13 showed better safety profile on HEK 293 cells than HepG2. SNEDDS of 4D showed higher antiproliferative activity than the 4D alone in both HepG2 and HEK 293 cells.
The aforementioned research demonstrated that the traditionally used Nigerian plant Annona Senegalensis has phytoconstituents which exhibit moderate antiplasmodial activity and has future potential for its extract and its fractions to be suitably formulated to enhance its biopharmaceutical properties.